The Sterile-20 (Ste20) family of the protein kinome includes both the p21-activated kinase (PAK) and germinal center kinase III (GCKIII) families. These proteins are critical for a variety o cellular functions including cell polarization and migration, cell cycle, apoptosis and axon outgrowth and regeneration. Dysregulation of Ste20 kinases is frequently observed in cancer and may be an important causative factor in cerebral cavernous malformations. Consequently, understanding the molecular basis for regulation of the Ste20 kinases is biologically and clinically important. In this proposal we will investigate the structural basis for normal and alteed regulation of two important sub-families of the Ste20 kinases, the type II PAKs and the GCKIII kinases, and will investigate the molecular level determinants of differences in specificity between the two sub-families. In Aim 1 we will investigate the regulation of type II PAKs. Type II PAKs encompass the proteins PAK4, PAK5 and PAK6. Whereas the molecular basis for how type I PAKs (PAK1, PAK2 and PAK3) are regulated by inter- molecular autoinhibition is well established, the type II PAKs are not regulated by the same mechanism. Type II PAKs do not include a conserved AID (autoinhibitory domain), and although some studies suggest that interaction of type II PAKs with GTPases is important for localization but not kinase activity, there is also intriguing data suggesting that the N-terminal region of the type II PAKs can play a role in autoregulation. Therefore, in Aim 1 we will investigate the molecular basis for type II PAK regulation. In Aim 2 we investigate the regulation of the GCKIII class of protein kinases: MST4, STK24 and STK25. The regulation of this family of protein kinases is not understood at the molecular level. However, each of these proteins uses a C-terminal region to directly interact with the cerebral cavernous malformation-associated protein CCM3 which may be a regulatory interaction. Therefore in Aim 2 we will determine the structural basis for GCKI association with CCM3, and CCM3-mediated modulation of GCKIII kinase activity. In Aim 3, we propose to probe the structural determinants of substrate selectivity by Ste20 kinases through a combination of X-ray crystallography and structure-guided mutagenesis. The Ste20 family includes closely related kinases having drastically different phosphorylation motifs, providing a unique and unprecedented opportunity to probe the structural requirements for substrate recognition through the introduction of exchange mutants. Overall, the studies proposed here will significantly improve our understanding of Ste20 kinase regulation and specificity, and will provide a better understanding of the atomic-level mechanisms that are disrupted in cancer and cerebral cavernous malformations.